Comparative analysis of three next-generation sequencing techniques to measure nosZ gene abundance in Missouri claypan soils.

Quantitative next-generation sequencing techniques have been critical in gaining a better understanding of microbial ecosystems. In soils, denitrifying microorganisms are responsible for dinitrogen (N2) production. The nosZ gene codes for nitrous oxide reductase, the enzyme facilitating the reduction of nitrous oxide (N2O) to N2. The objectives of this research were to: 1) understand how soil depth influences RNA concentration and nosZ gene abundance; 2) assess the spatial dependence of nosZ gene abundance in two claypan soil fields; and 3) compare and evaluate multiple RNA-based sequencing methods for quantifying nosZ gene abundance in soils in relation to dinitrogen (N2) production. Research sites consisted of two intensively studied claypan soil fields in Central Missouri, USA. Soil cores were collected from two landscape transects across both fields and analyzed for extractable soil RNA at two depths (0-15 cm and 15-30 cm). Measurements of nosZ gene abundance were obtained using real-time quantitative polymerase chain reaction (RT-qPCR), droplet digital polymerase chain reaction (ddPCR), and nanostring sequencing (NS). In both fields, soil RNA concentrations were significantly greater at 0-15 cm depth compared to 15-30 cm. These data indicated low overall soil microbial activity below 15 cm. Due to low quantities of extractable soil RNA in the subsoil, nosZ gene abundance was only determined in the 0-15 cm depth. Sequencing method comparisons of average nosZ gene abundance showed that NS results were constrained to a narrow range and were 10-20-fold lower than ddPCR and RT-qPCR at each landscape position within each field. Droplet digital PCR appears to be the most promising method, as it reflected changes in N2 production across landscape position.

Degradation kinetics of veterinary antibiotics and estrogenic hormones in a claypan soil.

Veterinary antibiotics and estrogens are excreted in livestock waste before being applied to agricultural lands as fertilizer, resulting in contamination of soil and adjacent waterways. The objectives of this study were to 1) investigate the degradation kinetics of the VAs sulfamethazine and lincomycin and the estrogens estrone and 17ß-estradiol in soil mesocosms, and 2) assess the effect of the phytochemical DIBOA-Glu, secreted in eastern gamagrass (Tripsacum dactyloides) roots, on antibiotic degradation due to the ability of DIBOA-Glu to facilitate hydrolysis of atrazine in solution assays. Mesocosm soil was a silt loam representing a typical claypan soil in portions of Missouri and the Central United States. Mesocosms (n = 133) were treated with a single target compound (antibiotic concentrations at 125 ng g-1 dw, estrogen concentrations at 1250 ng g-1 dw); a subset of mesocosms treated with antibiotics were also treated with DIBOA-Glu (12,500 ng g-1 dw); all mesocosms were kept at 60% water-filled pore space and incubated at 25 °C in darkness. Randomly chosen mesocosms were destructively sampled in triplicate for up to 96 days. All targeted compounds followed pseudo first-order degradation kinetics in soil. The soil half-life (t0.5) of sulfamethazine ranged between 17.8 and 30.1 d and ranged between 9.37 and 9.90 d for lincomycin. The antibiotics results showed no significant differences in degradation kinetics between treatments with or without DIBOA-Glu. For estrogens, degradation rates of estrone (t0.5 = 4.71-6.08 d) and 17ß-estradiol (t0.5 = 5.59-6.03 d) were very similar; however, results showed that estrone was present as a metabolite in the 17ß-estradiol treated mesocosms and vice-versa within 24 h. The antibiotics results suggest that sulfamethazine has a greater potential to persist in soil than lincomycin. The interconversion of 17ß-estradiol and estrone in soil increased their overall persistence and sustained soil estrogenicity. This study demonstrates the persistence of these compounds in a typical claypan soil representing portions of the Central United States.